This invention relates to a method of producing genetically transformed plants which are capable of expressing a desired gene product, and to clones of said genetically transformed plants having the same ability. More particularly, the plants concerned are of a type which produce a voluminous fluid and the target gene product is expressed in said fluid. Most preferably, the plants are rubber (Hevea) plants and the genes are foreign genes that code for pharmaceutically valuable protein products which can then be harvested in the latex produced by the plants and recovered therefrom.
Techniques for the genetic transformation of various microorganisms, such as yeasts, fungi and bacteria, for the purposes of producing specific proteins through the expression of "foreign" genes are well known. However, microorganisms require the maintenance of suitable conditions in which to survive and multiply. For example, the ambient temperature, pH value and aeration level usually need to be carefully controlled, while nutrients must be added to the culture medium in carefully regulated doses and waste products removed. Rigorous aseptic practices must be observed in order to avoid contamination by extraneous microbes. Microorganisms are thus normally cultured in sophisticated fermentors or bioreactors and which are housed in expensively maintained factories. Such overheads are reflected in the high price of the protein end-products.
More recently, attention has turned to the introduction of foreign genes into plants, such as tobacco plants. This application of genetic transformation techniques has allowed the incorporation of a variety of important genetic traits for crop improvement and also for the biotechnological production of extractable, valuable, foreign proteins (such as antibodies). Unlike microorganisms, plants tend to take care of themselves, requiring little more than sunlight, water and basic horticultural input, and can readily be cultivated on a cost efficient basis. Several different techniques have been developed to enable the introduction of foreign genes into various plant species and these include:
- the Agrobacterium vector system, which involves infection of the plant tissue with a bacterium (Agrobacterium) into which the foreign gene has been inserted. A number of methods for transforming plant cells with Agrobacterium are well known (Vancanneyt et al., 1990; Horsch et al., 1985; Bevan, 1984 and Herrera-Estrella et al., 1983); PA1 - the biolistic or particle gun method, which permits genetic material to be delivered directly into intact cells or tissues by bombarding regenerable tissues, such as meristems or embryogenic callus, with DNA-coated microparticles. The microparticles penetrate the plant cells, acting as inert carriers of the genetic material to be introduced (Gordon-Kamm et al., 1990 and Sanford et al., 1987). Microprojectile bombardment of embryogenic suspension cultures has proven successful for the production of transgenic plants of yellow-poplar (Dayton et al., 1992) cotton (Finer and McMullen, 1990), maize (Gordon-Kamm et al., 1990) and soybean (McMullen and Finer, 1990). Various parameters that influence DNA delivery by particle bombardment have been defined (Klein et al., 1988; Wang et al., 1988); PA1 - by imbibition of the foreign gene into the plant tissue (Simon, 1974); and PA1 - electroporation. PA1 i) inserting into the plant tissue a gene or gene fragment controlling the expression of a target product, and PA1 ii) regenerating a plant from said tissue, the genetically transformed plant being capable of expressing the target product in the fluid that it produces. PA1 i) harvesting the fluid from a genetically transformed fluid-producing tree or plant, or a clone thereof, and PA1 ii) recovering the target protein or other product from said fluid. PA1 - the Agave plant. Some varieties can produce up to a liter of exudate a day from their hollowed-out pith; PA1 - the coconut palm. Its inflorescence can be tapped to induce a copious flow of exudate; PA1 - plant organs that do not exude but nevertheless retain considerable volumes of fluid. For instance, the liquid endosperm of the young coconut fruit (i.e. the "water" or "milk") can be harvested in large quantities. PA1 i) The process of genetic transformation of the plant, whereby the DNA molecule representing the gene that codes for the target protein or other product is inserted into the genetic complement of tissues of the plant. It will be appreciated that the gene will also need to be accompanied by a promoter. While so-called "universal" promoters (which are not tissue specific and generally turn on gene expression in all tissues of the plant, including the sap or fluid) may be employed, the promoter is more preferably fluid-specific. In the case of the rubber plant, the promoter is most preferably latex-specific. PA1 ii) The regeneration of a plant from the transformed tissue, the planting out and nurturing to maturity. For example, through the technique of tissue culture the transformed callus tissue of Hevea is regenerated, via an embryo stage, into a plantlet which is transgenic and bears the inserted gene in its genetic complement. In time, the plantlet matures into a full grown transgenic rubber tree and the target protein or other product expressed by the inserted gene is present in its latex. PA1 iii) The harvesting of the target protein or other product in the fluid produced by the plant or tree and recovering the same therefrom. In the case of Hevea, the fluid harvested will of course be latex. Once the transgenic rubber plant has grown to sufficient maturity, usually after from two to five years, it is tapped and the latex harvested at regular intervals. The latex is then usually centrifuged so as to separate out the natural rubber, the aqueous C-serum and the so-called "bottom fraction". The target protein or other product may be contained in any part of the latex and can be extracted and purified by conventional means (such as preparative column chromatography). Preferably, it is present in the C-serum or in the bottom fraction, the latter comprising mainly lutoids and which are membrane-bound vesicles containing their own serum (B-serum). The B-serum can be released from lutoids by rupturing their membranes using detergents, such as Triton X-100, or by alternate freezing and thawing. Any of the target protein or other product that may be bound to biological membranes in the latex can be recovered by solubilising with detergents such as sodium dodecyl sulphate. PA1 (a) Insulin for diabetes treatment PA1 (b) Blood-clotting factors for haemophilia treatment PA1 (c) Blood clot-dissolving activators for cardiac treatment PA1 (d) Tumor necrotic factor for cancer treatment PA1 (e) Erythropoietin for anaemia treatment PA1 (f) Viral coat proteins for vaccine production PA1 (g) `PHB`, a plastic similar to polypropylene and which is used for the manufacture of bottles, wrappings, etc. PA1 - Production is continual, since the latex containing the target protein or other product is harvested at regular intervals, and product recovery is simple. As latex is a fluid, recovery of the target product does not involve tissue homogenisation. PA1 - The approach is environment-friendly. The process is driven by the sun and is thus energy efficient and essentially pollution-free. PA1 - Rubber trees require no special attention beyond routine horticultural maintenance. Their use is thus highly cost-efficient. PA1 - The technique is applicable for use for a wide range of target products. PA1 - The latex that flows out of the rubber tree is completely free of bacteria and animal viruses. This is in contrast to the rigorous aseptic practices which need to be adopted when genetically transformed microorganisms or animals are employed. PA1 - Rubber trees are amenable to clonal or vegetative propagation, most commonly by bud grafting. Other approaches include taking a cutting, marcotting or tissue culture. Thus, an unlimited number of genetically identical plants (clones) can be generated from a single transgenic plant--all of which will go on to express the desired product in their latex. PA1 - Rubber trees have an economic life of about thirty years. During this time, in addition to producing the target product, the genetically transformed tree will of course continue to produce natural rubber in its latex and which is a valuable commodity in its own right. When it is eventually felled, the tree also yields a valuable tropical timber (rubberwood) which is much sought after for export and furniture manufacture.
When this approach is adopted, however, the recovery of the target product involves the harvesting and destruction of the entire plant or at least a substantial portion of the plant. Even where the plant is not totally destroyed, it then requires a lengthy period of recovery for re-growth before a further harvest is possible. In addition, the extraction of the protein product from plants such as tobacco (and indeed also from most of the microorganisms which are commonly used for such purposes) involves the homogenization of tissue solids. This can be a relatively problematic and inefficient operation.
The Brazilian rubber tree, Hevea brasiliensis Muell-Arg, belongs to the family Euphorbiaceae and has been commercially exploited for the production of natural rubber for about a century. There are in fact nine species in the genus Hevea, but Hevea brasiliensis is the most widely cultivated and commercially valuable since it gives the highest yield of latex.
Latex is traditionally extracted from rubber trees by a method known as "tapping". This can be achieved either by a bark excision technique, in which a strip of bark is cut out of the tree trunk so as to initiate latex flow (subsequent tappings being carried out by excising a thin layer of bark from the same cut), or by a bark incision technique, according to which one or more punctures are made into the bark to initiate the flow of latex. Tapping is thus a non-destructive method of latex recovery and which may be conducted repeatedly and at regular intervals, typically every alternate day. Natural rubber constitutes about a third of the latex and can be readily extracted by various separation techniques, such as centrifugation.